mTOR is a serine/threonine kinase that is expressed ubiquitously. The mTOR enzyme is present in two cellular complexes, TORC1 and TORC2. mTOR was discovered and named based on its interaction with the drug rapamycin. This compound is an allosteric inhibitor that partially blocks TORC1 activity and generally does not inhibit TORC2 function. Rapamycin treatment of T and B lymphocytes profoundly blocks proliferation triggered by antigen and other mitogens, and rapamycin (sirolimus; Rapamune(R)) is an FDA-approved immunosuppressant. However, rapamycin and analogs have lesser effects on proliferation in other cellular systems including cancer cells. Although the effects of rapamycin on lymphocytes have been studied for 20 years, it remains unclear why the compound induces a profound block of proliferation selectively in lymphocytes. Paradoxically, a novel class of ATP-competitive mTOR kinase inhibitors (TOR-KIs) causes less immunosuppression than rapamycin despite causing more complete inhibition of both TORC1 and TORC2. The goal of this project is to define mechanisms by which rapamycin selectively blocks proliferation of normal lymphocytes. Based on preliminary data, we propose two hypotheses that form the basis of separate specific aims. Aim 1 will test the hypothesis that rapamycin causes more complete and sustained inhibition of S6 kinases, key substrates of TORC1. There are two S6 kinases, S6K1 and S6K2, but their roles in lymphocyte activation and proliferation have not been reported. We will use a novel mouse model that will allow a chemical genetic approach to specifically inhibit S6K activity in normal T and B cells. We will use this model to compare the effects of S6K inhibition with the effects of rapamycin and TOR-KIs. Aim 2 will test the hypothesis that rapamycin inhibits kinase-independent functions of mTOR. We will generate a novel mouse strain in which kinase-dead mTOR can be expressed in a cell-specific manner. This will allow us to test the prediction that lymphocytes expressing kinase-dead mTOR will retain residual proliferation that remains sensitive to rapamycin. We will also begin a candidate approach to test possible kinase-independent mTOR functions.